Language selection

Search

Patent 2746288 Summary

Third-party information liability

Some of the information on this Web page has been provided by external sources. The Government of Canada is not responsible for the accuracy, reliability or currency of the information supplied by external sources. Users wishing to rely upon this information should consult directly with the source of the information. Content provided by external sources is not subject to official languages, privacy and accessibility requirements.

Claims and Abstract availability

Any discrepancies in the text and image of the Claims and Abstract are due to differing posting times. Text of the Claims and Abstract are posted:

  • At the time the application is open to public inspection;
  • At the time of issue of the patent (grant).
(12) Patent Application: (11) CA 2746288
(54) English Title: DETECTION OF IFI16 IN BODY FLUIDS
(54) French Title: DETECTION DE IFI16 DANS DES FLUIDES CORPORELS
Status: Deemed Abandoned and Beyond the Period of Reinstatement - Pending Response to Notice of Disregarded Communication
Bibliographic Data
(51) International Patent Classification (IPC):
  • G1N 33/564 (2006.01)
(72) Inventors :
  • MONDINI, MICHELE (Italy)
  • LANDOLFO, SANTO (Italy)
  • GARIGLIO, MARISA (Italy)
  • COSTA, SILVIA (Italy)
  • MIRAGLIA, ERICA (Italy)
  • GUGLIESI, FRANCESCA (Italy)
(73) Owners :
  • NOTOPHARM SRL
(71) Applicants :
  • NOTOPHARM SRL (Italy)
(74) Agent: NORTON ROSE FULBRIGHT CANADA LLP/S.E.N.C.R.L., S.R.L.
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 2009-12-14
(87) Open to Public Inspection: 2010-06-17
Availability of licence: N/A
Dedicated to the Public: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/EP2009/067128
(87) International Publication Number: EP2009067128
(85) National Entry: 2011-06-08

(30) Application Priority Data:
Application No. Country/Territory Date
61/122,091 (United States of America) 2008-12-12

Abstracts

English Abstract


The present invention relates to methods for the qualitative and/or
quantitative determination of interferon
inducible protein 16 (I Fl 16) in an extracellular form.


French Abstract

La présente invention porte sur des procédés pour la détermination qualitative et/ou quantitative de la protéine 16 inductible par interféron (I F1 16) sous forme extracellulaire.

Claims

Note: Claims are shown in the official language in which they were submitted.


-1-
claims
1. An in vitro method for determining extracellular interferon inducible pro-
tein 16 (IFI16) in a sample.
2. The method of claim 1, wherein the sample is a body fluid sample, e.g.,
blood, plasma or serum, or a supernatant of a tissue sample or a su-
pernatant of a cell culture sample.
3. The method of claim 1 or 2, wherein the determination comprises:
(a) contacting the sample with at least one receptor, which
specifically binds to IFI16, and
(b) detecting the specific binding of the receptor to IFI16.
4. The method of claim 3, wherein the sample is contacted with at least
two receptors, which specifically bind to IFI16, wherein one of the re-
ceptors is a detectable receptor and the other receptor is immobilized
on a solid phase or carries a solid-phase binding group.
5. The method of claim 3 or 4, wherein at least one receptor is an anti-
body or an antigen-binding fragment thereof.
6. The method of claim 4 or 5, wherein the detectable receptor carries a
detectable labelling group, e.g. an enzymatic, fluorescent, radioactive
or nucleic acid labelling group.
7. The method of any one of claims 1-6, wherein the sample is a human
sample.

-2-
8. The method of any one of claims 1 to 7, wherein the presence and/or
an increased amount of extracellular IFI16 is indicative for a pathologic
condition.
9. The method of any one of claims 1-7, wherein the presence and/or an
increased amount of extracellular IFI16 is indicative of an autoimmune
and/or inflammatory disorder.
10. The method of claim 9, wherein the autoimmune disorder is selected
from Systemic Sclerosis (SSc), Systemic Lupus Erythematosus (SLE)
and Sjogren's Syndrome (SjS).
11. The method of claim 9, wherein the disorder is selected from rheumat-
oid arthritis.
12. The method of any one of claims 1-7, wherein the presence and/or an
increased amount of extracellular IFI16 is indicative of an infective dis-
order.
13. The method of claim 12, wherein the infective disorder is HCV infection.
14. The method of any one of claims 1-13, further comprising determining
of at least one additional diagnostic marker.
15. The method of claim 14, wherein the at least one additional diagnostic
marker is an anti-IFI16-autoantibody.

Description

Note: Descriptions are shown in the official language in which they were submitted.


CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-1-
Detection of 1171116 in body fluids
Specification
The present invention relates to methods for the qualitative and/or
quantitative determination of interferon inducible protein 16 (IF116) in an
extracellular form.
The interferon-inducible protein IF116 belongs to the family of Interferon
(IFN)-activatable genes designated HIN200 in the human and Ifi200 in the
murine species. It was demonstrated that IF116 is a nuclear phosphoprotein
which participates in the inhibition of cell cycle progression, and
involvement
of the inflammatory process.
Immunohistochemical analysis of IF116 expression in normal human tissues
revealed that it is expressed in a highly restricted pattern in selected cells
within certain organs. IF116 is expressed in CD34+ myeloid precursor cells
and remains strongly expressed within monocyte precursors, peripheral
blood monocytes, and throughout lymphoid development. IF116 was found in
epithelial cells of the skin, gastrointestinal tract, urogenital tract and
glands
and ducts of breast tissues. In addition, all vascular endothelial cells from
both blood and lymph vessels strongly expressed IFI16.
IFI16 expression can be induced by interferons, as well as by an array of
cytokines and differentiating agents. In HL-60 cells, IFi16 was induced by
dimethylsulfoxide, retinoic acid, and 1,25 dihydroxy vitamin D3. IF116 is
stimulated in HUVEC endothelial cells by oxidative stress and by pro-
inflammatory molecules such as TNF-a and interleukin-1 R (IL-1(3).
Several lines of evidence link the interferons (IFNs) to autoimmune
disorders, in particular to SSc and Systemic Lupus Erythematosus (SLE).
Many observations suggest a role for IFI16 in systemic autoimmune
diseases, in which chronic inflammation is involved. IF116 expression was

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-2-
greatly increased and ubiquitously detected in all layers of the epidermis in
the lesional skin from both SSc and SLE patients. Furthermore, the dermal
inflammatory infiltrate showed IF116 positive staining, indicating that it is
expressed at a high level in lymphocytes, fibroblasts and EC.
Further, autoantibodies against IF116 were found in the sera of patients
affected by Systemic Sclerosis (SSc), Systemic Lupus Erythematosus (SLE)
and Sjogren's Syndrome (SjS).
A possible use of IF116 as molecular marker, however, appeared to be
limited to solid tissue samples, since previously described results show that
the protein IF116 has an intracellular localization. Therefore, previous
detection procedures were carried out with solid tissue samples from
patients. Taking solid tissue samples from lesional tissue has, however,
disadvantageous effects for the patients and is difficult. Moreover, since it
is
difficult to obtain solid tissue samples from healthy subjects, the use of
IF116
as molecular marker has been limited by the difficulty to compare IF116
expression in lesional vs healthy samples.
Until now, experiments to evaluate possible IF116 dysregulation in
pathological conditions were carried out on solid tissue samples or cultured
cells and to extracts from tissues, circulating or cultured cells, since it
was
assumed in the prior art that IF116 is a protein which is active within the
cell
and is located in the nucleolus and the nucleoplasm of human cells (as
shown by both confocal microscopy and immunoblotting of nuclear proteins).
As a mechanism for the generation of anti-IF116 autoantibodies, it was
hypothesized in the literature that IF116 could be released from dying cells
(Mondini M. et at., 2006). The process of cell death is recognized as a
possible source of several autoantigens, and the most accepted mechanism
of release is the relocalization of the nuclear antigens in apoptotic blebs
(thus restricted within the membrane barrier) and/or their exposure to
immune effectors at cell-membrane levels. This phenomenon has been

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-3-
demonstrated for several autoantigens, including Ro/SSa, La/SSB and
oxidized nuclear antigens (i.e. LeFeber et al., 1984; Casciola-Rosen L. et
al.,
1994; Saegusa et al. 2002). An association of extracellular IF116 protein with
pathological conditions has not been published.
Based on this state of knowledge, however, it could not have been expected
that a detectable amount of extracellular form of IF116 might exist in the
extracellular environment and that such amount of an extracellular IF116
would be associated with pathological conditions.
Surprisingly, however, the present inventors have found that extracellular
IF116 may be determined in appropriate samples by simple and direct
analytic methods.
Thus, in a first aspect, the present invention refers to an in vitro method
for
determining extracellular interferon inducible protein 16 (IF116) in a sample.
In particular, the present inventors found that the presence and/or an
increased amount of extracellular IF116 is indicative for a pathological
condition. Thus, the present invention is suitable as a diagnostic method for
any pathologic condition associated with increased presence of IF116
compared to a normal, i.e. healthy, control.
The term "sample" as used herein refers to a biological sample obtained
from the purpose of evaluation in vitro. In the methods of the present
invention, the sample, which is tested for extracellular IF116, is preferably
a
body fluid sample, e.g. blood, plasma, serum, urine, saliva etc.
Alternatively,
the supernatant of a tissue sample or the supernatant of a cell culture
sample may be tested. Preferably, the sample preparation does not involve
any lysis of cells, particularly no lysis of cells known to express IF116,
e.g.
epithelial or endothelial cells. The sample may be derived from a mammalian
organism, e.g. a human organism or a mammalian, e.g. human cell culture
according to known methods.

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-4-
In the context of the invention, the term "body fluids" comprises all kinds of
body fluids, optionally diluted or concentrated. Examples are blood, serum,
plasma, amniotic fluid, brain/spinal cord fluid, liquor, cerebrospinal fluid,
sputum, throat and pharynx secretions and other mucous membrane
secretions, synovial fluids, ascites, tear fluid, lymph fluid and urine.
Preferably, the body fluid is blood, plasma or serum.
According to the present invention, the detection of IF116 may include the
detection of full-length IF116, or fragments thereof, particularly fragments,
which have immunological activity of IF116, e.g. which are immunologically
detectable, and which may be produced by cleavage, e.g. enzymatic
cleavage, and which may be indicative of pathological conditions, e.g.
inflammatory diseases.
The term "determination" and/or "detection" comprises a qualitative or a
quantitative determination of extracellular IF116 in a sample. In a preferred
embodiment, the determination is a qualitative or semi-quantitative determin-
ation, i.e. it is determined whether IF116 is present or absent or it is
determ-
ined whether the concentration of IFI16 is above or below a cut-off value. As
the skilled artisan will appreciate, in a Yes-(presence) or No-(absence) as-
say, the assay sensitivity is usually set to match the cut-off value. A cut-
off
value can, for example, be determined from the testing of a group of healthy
individuals. Preferably, the cut-off is set to result in a specificity of 90%,
also
preferred the cut-off is set to result in a specificity of 95%, or also
preferred
the cut-off is set to result in a specificity of 98%. Presence of a value
above
the cut-off value can, for example, be indicative for the presence of patholo-
gical conditions, in particular for example autoimmune and/or inflammatory
disorders. In a further preferred embodiment, the determination is a quantit-
ative determination. In this embodiment, the concentration of extracellular
IF116 is correlated to underlying diagnostic question like, e.g. stage of dis-
ease, disease progression or response to therapy.

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-5-
Preferably, the determination of extracellular IF116 comprises:
(a) contacting the sample with at least one receptor, which
specifically binds to IFI16, and
(b) detecting the specific binding of the receptor to IFI16.
According to the invention, the term "specific binding" describes a specific
interaction between a receptor and IF116 or a fragment thereof. The specific
interaction can be characterised with a "key-lock-principle". The receptor and
IF116 have structures or motifs which fit with each other specifically, as
e.g.
an antigenic determinant (epitope) which interacts with the antigen binding
site of an antibody.
The receptor, which specifically binds to IF116, has at least an affinity of
1061/mol for IF116, preferably of at least 10' I/mol for IF116, more
preferably
an affinity of at least 10$ I/mol or also preferred of at least 109 I/mol for
IF116.
As the skilled artisan will appreciate the term specific is used in particular
to
indicate that other biomolecules present in the sample do not significantly
bind to the receptor specific for IFI16. Preferably, the level of binding to a
biomolecule other than the target IF116 results in a binding affinity which is
at
most only 10% or less, only 5% or less only 2% or less or only 1 % or less of
the affinity to the target IF116, respectively. A preferred receptor, which
specifically binds to IF116, will fulfill both the above minimum criteria for
affinity as well as for specificity.
In a further preferred embodiment of the method according to the invention,
the detection of a specific binding of IF116 with the first receptor in step
(a),
the sample is contacted with a second receptor for lF116, which binds with
an epitope of IF116 and which is accessible after binding of the first
receptor
with IF116.
In an especially preferred embodiment, the method of the invention involves
the use of at least two receptors which specifically bind to IFl16, wherein
one
receptor is a detectable receptor and the other receptor is immobilized on a

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-6-
solid phase or carries a group, which allows immobilization to a solid phase,
e.g. via specific binding to a complementary member of a binding pair on the
surface.
This preferred embodiment relates, for example, to methods taking
advantage of the mechanistic principle of the sandwich ELISA. This principle
is generally known to the person skilled in the art. Furthermore, a
corresponding method is described in Examples 1 and 3.
The detectable receptor may carry a detectable labelling group. Methods
allowing labelling of a receptor are known in the art. Alternatively, the
detectable receptor group may be specifically recognized by means of
another, third receptor comprising a detectable labelling group.
Preferred examples of such labelling groups are radioactive or fluorescent
labelling groups.
Further preferred labelling groups comprise enzyme labelling groups, e.g.
alkaline phosphatase, peroxidase, [beta]-galactosidase, glucoamylase,
urease and chloramphenicol acetyltransferase. Appropriate examples and
the use of necessary substrates for the detection by means of enzymatic
reactions are known to the person skilled in the art, amongst others from the
package leaflet of commercially available detection kits. Such commercially
available kits often contain antibodies which recognise the antibodies of
specific species, e.g. anti-mouse, and to which enzymes emitting signals are
coupled. Thus, corresponding antibodies are examples of the third receptor,
which recognise a specific labelling of the second receptor, that is its Fc
part.
The receptor may be selected from the group consisting of peptides,
polypeptides, low-molecular substances, antibodies or fragments or
derivatives thereof and aptamers. In a preferred embodiment, the receptor is
an antibody or an antigen binding fragment thereof.

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-7-
The term "peptide" usually refers to amino acid chains with up to 30 amino
acids.
The term "polypeptide" refers to peptides which usually comprise more than
30 amino acids and includes proteins.
The term "low-molecular substance" or small molecule refers to molecules
which are of lower molecular complexity than the macromolecules defined
above. In the literature, the term "low-molecular substance" is not used in a
uniform manner. In WO 89/03041 and WO 89/03042, molecules with a
molecular mass of up to 7000 g/mol are described as small molecules.
Usually, however, molecular masses between 50 and 3000 g/mol, more
often, however, between 75 and 2000 g/mol and mostly in the range
between 100 and 1000 g/mol are stated. Examples are known to the person
skilled in the art from the documents (W086/02736, W097/31269, U.S. Pat.
No. 5,928,868, U.S. Pat. No. 5,242,902, U.S. Pat. No. 5,468,651, U.S. Pat.
No. 5,547,853, U.S. Pat. No. 5,616,562, U.S. Pat. No. 5,641,690, U.S. Pat.
No. 4,956,303 and U.S. Pat. No. 5,928,643. Low-molecular substances can
be of organic or inorganic nature.
According to the invention, the term "antibody" comprises polyclonal sera as
well as monoclonal antibodies.
Monoclonal antibodies and methods for the production thereof are known to
the person skilled in the art. These are based on a method first described by
Kohler and Milstein (1975). This method is described in detail in, amongst
others, the laboratory manual by Harlow and Lane (Antibodies, A laboratory
manual; Cold Spring Harbor Laboratory; (1988); Chapter 6). By this
definition, bispecific antibodies, synthetic antibodies and fragments or
derivative of these antibodies are also comprised. These comprise
fragments such as Fab, Fv or scFv and chemically modified derivatives of
these antibodies or antibody fragments.

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-8-
Aptamers are, in principle, known to the person skilled in the art from prior
art.
Preferably, the method according to the invention is an ELISA, an EIA or a
RIA. Appropriate methods are, in principle, known to the person skilled in the
art from Harlow and Lane, loc. cit. and Rehm, loc. cit.
The surprising result that IFI16, so far known as an intracellular protein, is
found in extracellular environment makes it possible to analyse IF116 in the
culture supernatant of tissue samples, samples of body fluids or samples of
cell culture supernatants. By means of the method according to the
invention, IF116 can be detected in a simple and fast manner and, thus,
serves as diagnostic parameter.
An association of extracellular IF116 with pathological condition has not been
published yet. Thus, there is no suggestion that a determination of
extracellular IF116 in body fluids would allow assessment of a pathological
condition. Surprisingly, it was found in the present invention that a
determination of the presence and/or amount of extracellular IF116 in body
fluid samples allows the assessment of pathological conditions, in particular
autoimmune and/or inflammatory disorders. In particular, the inventors found
out that a reliable assessment of these pathological conditions is possible by
measuring IFI16 within an extracellular liquid sample from an individual, i.e.
no tissue and no biopsy sample is required in diagnosis of the disease when
using extracellular IF116 protein as marker. Even more unexpectedly it was
found that an increased level of extracellular IF116 as measured from bodily
fluid of an individual is associated with autoimmune or inflammatory
disorders.
On the basis of the surprising result that IF116 is significantly present in
body
fluids from SSc, SLE, SjS and rheumatoid arthritis (RA) patients, while it is
only barely detectable in body fluids from healthy subjects, a particular
usefulness as diagnostic tool for autoimmune disorders is assigned to the

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-9-
method according to the invention.
A specific role in the onset of inflammation has been assigned to IFI16 that,
when overexpressed in endothelial cells, upregulates the expression of
several proinflammatory cytokines and is involved in TNF-a and IFN
signaling.
Thus the detection of IFI16 in the body fluid of patients by means of the
method according to the invention is also particularly important with respect
to inflammatory diseases, including autoimmune disorders and possibly to
bacterial and viral infectious diseases (AIDS, meningitis, HCV infections),
allergies, transplant reactions, cardiovascular and tumour diseases and so
on. Furthermore, these are important for the determination of the response
reaction with patients under treatment with inflammatory cytokines (e.g.
interferon-a).
The detection or the quantification of IF116 in a sample of a body fluid of a
patient allows conclusions to be drawn about some clinical features of the
patient.
Methods for obtaining the samples mentioned are known to the person
skilled in the art. Optionally, the method according to the invention,
moreover, comprises one or several washing steps prior to or after each
method step. These washing steps serve the minimization of unspecific
reactions (false positive or false negative detection) and can improve the
sensitivity of the method. Suitable washing buffers and their composition are,
in principle, known to the person skilled in the art. Physiological buffer
solutions are preferred.
A preferred embodiment of the method according to the invention, moreover,
comprises step (a') or (a") prior to contacting with the first receptor: (a')
labelling of the proteins contained in the sample; or (a") labelling of the
first
receptor.

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-10-
The proteins contained in the sample and/or the first receptor can, for
example, be labelled chemically, e.g. by coupling of labelled chemical groups
or markers to free amino groups of cysteines contained in the proteins.
Examples of such marked chemical groups are groups containing special,
detectable radioisotopes. For example fluorescent dyes can also serve as
markers. A further example of appropriate markers are nucleic acids. The
presence of proteins or receptors in a sample which are labelled in such a
way can then be detected with suitable primers in a polymerase chain
reaction (PCR).
Furthermore, it is possible to label proteins physiologically, i.e. by the
metabolic integration of labelled molecules. For this purpose, cells are, for
example, incubated with radioactively labelled metabolites. Proteins
originating from the biosynthesis of these cells during this incubation period
and in which the labelled metabolites were integrated are marked. This
method is e.g. suitable to label antibodies secreted by cells which produce
antibodies.
In a further preferred embodiment of the method according to the invention,
the receptor is immobilised on a surface prior to contacting with a sample
suspected to contain IF116.
According to an alternative embodiment of the method of the invention, the
receptor is immobilised on a surface after contacting with a sample
suspected to contain IFI16.
Receptors can be immobilised in various way. The appropriate method
depends on various factors, such as e.g. the type of receptor or the material
of the surface. An immobilisation can take place covalently or by adsorption.
According to a preferred embodiment of the method according to the
invention, the receptors are proteins, particularly preferred antibodies. Also
preferred is the use of peptides or organic molecules as receptors.

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-11-
For the immobilisation of receptors which are proteins, methods are
described in which the receptors are immobilised directly on a surface by
means of passive adsorption. Normally, an appropriate surface consists of a
polymer plastic material (e.g. polystyrene, polyvinyl, latex) and e.g. in form
of
microtitre plates or multi-well plates, membranes or spheric "beads" (cross-
linked polymers in particle form) are used for this purpose (Lowman, Annu.
Rev. Biophys. Biomol. Struct. 26 (1997), 401-24).
In a further preferred embodiment of the method according to the invention,
the material of the surface is selected from the group consisting of
sepharose, latex, glass, polystyrene, polyvinyl, nitrocellulose and silicon.
Further preferred, the surface in the method according to the invention is a
membrane, a bead, a chip or a plate.
Examples of beads are sepharose beads or latex beads, to which, optionally,
ligands are bound, which promote the immobilisation of the receptors to the
surface. Such ligands are, for example, protein A or protein G which promote
a binding of antibodies to a surface via the Fc part of the antibody. The
binding of the receptor to a carrier material can also be achieved by a
covalent chemical coupling reaction (e.g. hydrazide coupling). Another
example of the immobilisation of the receptors to the surface by means of
ligands is the use of biotin and avidin or streptavidin.
Examples of chips are silicon plates onto which a plurality of different or
the
same receptors can be immobilised systematically. This allows the analysis
of a plurality of different parameters in a sample or the analysis of a
plurality
of different samples as to one or several parameters, e.g. identification
and/or quantification of IF116 or fragments of this protein in different
tissue
samples, samples of body fluid or samples of cell culture supernatants.
Examples of the plates mentioned are microtitre plates or multi-well plates.
Preferably, these have 6, 12, 24, 48, 96, 128, 356, 1024 or more wells. In

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-12-
Example 1, a method is described wherein 96-well plates are used.
According to a further preferred embodiment of the method, it further
comprises step (b') prior to the step of detection of a specific binding: (b')
Precipitating the beads with the complexes of the first receptor and IFI16.
Beads can be precipitated from a sample e.g. in a gravimetric manner. This
can be accelerated, for example, by centrifugation. Appropriate methods are
known to the person skilled in the art, amongst others from Rehm, Der
Experimentator: Proteinbiochemie/Proteomics, Spektrum Akademischer
Verlag, 2002.
In a further preferred embodiment of the method according to the invention,
the detection of the specific binding between a receptor and IF116
comprises a gel electrophoretic separation of the sample and, optionally,
furthermore, a Western blot analysis. Appropriate methods are known to the
skilled person, among others from Rehm, loc. cit. Furthermore, a
corresponding method is described in Examples 2 and 4.
The method according to the invention is preferably carried out
automatically. This is possible, amongst others, by the use of pipetting
robots and for an automated analysis of optimised processes.
Furthermore, the invention refers to the use of body fluids or a sample of a
cell culture supernatant, as defined above, for the detection of extracellular
IFI16. Preferably, the positive detection is indicative for the presence of a
pathological condition, in particular an inflammatory and/or autoimmune
disease.
Further, the method of the invention also comprises the determination of at
least one additional diagnostic marker, e.g. a diagnostic marker indicative of
an autoimmune and/or inflammatory disorder. In a preferred embodiment,
the at least one additional diagnostic marker is an anti- IF116-autoantibody.

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-13-
The determination of several diagnostic markers may be carried out in
parallel on a single sample or different aliquots of a single sample or on
different samples. The concentration of the diagnostic markers are then
interpreted independently, e.g. using an individual cut-off value for each
marker, or they are combined for interpretation.
Finally, the present invention refers to a reagent kit for diagnostic use
comprising:
(i) at least one receptor, which specifically binds to IF116, and
(ii) further kit components, e.g. buffers, salts, reagents and/or instructions
for use.
Preferred embodiments of reagent kits comprise two receptors, wherein one
receptor is a detectable receptor and one receptor is an immobilized or an
immobilizable receptor.
Further, the present invention shall by explained in more detail by the follow-
ing figures and examples without being limited thereto.
Figures
Fig. 1: Schematic representation of a IF116 sandwich ELISA.
Fig. 2: Sensitivity and linearity of IF116 sandwich ELISA.
ELISA microtitre plates were coated with a polyclonal rabbit-anti-IF116
antibody. Subsequently, the plates were washed with PBS-Triton (PBS-T;
0.25% Triton X100 in PBS) and for 30 minutes, free binding sites were
saturated with PBS-T/BSA 3% (PBS-TB) at 37 . After washing with PBS-T,
an incubation followed (1 h) with purified 6His-IF116 protein, diluted in 5%
FCS in PBS-T was used as standard. BSA served as negative control. The
samples were washed 3 times with PBS-T, in each case monoclonal mouse
anti-IF116 antibody was added and incubated for 1 h at room temperature.
After washing four times with PBS-T, an incubation followed (1 h, room

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-14-
temperature) with 100 pl, in each case, of HRP-conjugated anti-mouse
antibody diluted in PBS-TB. After 3 washing steps, the IF116 protein/antibody
complex was visualised by incubation with tetramethylbenzidine (TMB) and
stopped with Stop Solution. The absorption was measured at 450 nm in the
micro plate reader. The determination of the concentration was carried out
using the standard curve of Figure 2 for which increasing concentrations of
purified 6His-IF116 were used. The linearity of the measurement is indicated
for a range of 1 to 15.6 ng/ml .
FIG.3: Measuring circulating IF116 in autoimmune patients and healthy
subjects.
The concentration of circulating IF116 in sera was determined by means of
ELISA in patients suffering from SSc (99), SLE (30), SjS (20), RA (30) and
patients with hepatitis C virus infection (HCV, 30) in healthy subjects
(CTRLS, 54).
ELISA microtitre plates were coated with a polyclonal rabbit-anti-IF116
antibody. Subsequently, the plates were washed with PBS-Triton (PBS-T;
0.25% Triton X100 in PBS) and for 30 minutes, free binding sites were
saturated with PBS-T/BSA 3% (PBS-TB) at 37 . After washing with PBS-T,
an incubation followed (1 h) with 5 pl of different sera samples in a final
volume of 100 pl. Purified 6His-IFI16 protein, diluted in 5% FCS in PBS-T
was used as standard. BSA served as negative control. The samples were
washed 3 times with PBS-T, in each case monoclonal mouse anti-IF116
antibody was added and incubated for 1 h at room temperature. After
washing four times with PBS-T, an incubation followed (1 h, room
temperature) with 100 pl, in each case, of HRP-conjugated anti-mouse
antibody diluted in PBS-TB. After 3 washing steps, the IF116 protein/antibody
complex was visualised by incubation with tetramethylbenzidine (TMB) and
stopped with Stop Solution. The absorption was measured at 450 nm in the
micro plate reader. The determination of the concentration was carried out
using the standard curve of Figure 2 for which increasing concentrations of
purified 6His-IF116 were used. The linearity of the measurement ranged from

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-15-
20 to 400 ng/ml IF116 in the sera. Sera with a concentration outside the
linearity range (<20 ng/ml or >400 ng/ml) are plotted as having 0 ng/ml or
400 ng/ml respectively.
The IF116 serum protein was detectable in a fraction of patients sera
(ranging from 54% to 84%), while IF116 serum concentration was below the
detection limit of the assay in all the healthy subjects.
FIG.4: Identification of extracellular IF116 in cell supernatants.
Human keratinocytes were exposed to UVB irradiation at a dose of 200, 400
or 800J/m2 (UV 200, UV 400 or UV 800 respectively) or mock irradiated
(NT) and then incubated for 16 or 24 hours (16h or 24h respectively).
Supernatants were collected and extracellular proteins precipitated by TCA
as described in Example 2. Immunoblotting analysis using anti-IFI16
polyclonal antibodies revealed the presence of extracellular IF116 in
supernatants of cells exposed to UVB irradiation doses of 400 and 800 J/m2.
Total cellular proteins extracted from human keratinocytes (TE), expressing
intracellular IFI16, were used as a positive control for IF116 immunoblotting.
Fig. 5: Sensitivity and linearity of IFI16 sandwich ELISA with improved
linearity.
ELISA microtitre plates were coated with a polyclonal rabbit-anti-IF116
antibody. Subsequently, the plates were washed and free binding sites were
saturated with PBS/0,05%Tween-20/3%BSA (PBS-TB) at room temperature
for 1 hour. After washing, an incubation followed (1 h, room temperature)
with purified 6His-IF116 protein, diluted in 5% FBS in PBS/0,05%Tween-
20/1%BSA (PBS-TD), that was used as standard. BSA served as negative
control. The samples were washed and in each case monoclonal mouse
anti-IFI16 antibody was added and incubated for 1 h at room temperature.
After washing, an incubation followed (1 h, room temperature) with HRP-
conjugated anti-mouse antibody. After washing, the IF116 protein/antibody
complex was visualized by incubation with tetramethylbenzidine (TMB) and
stopped with Stop Solution. The absorption was measured at 450 nm in the

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-16-
micro plate reader. The determination of the concentration was carried out
using the standard curve of Figure 5 for which increasing concentrations of
purified 6His-IF116 were used. The linearity of the measurement is indicated
fora range of 1 to 32 ng/ml .
Fig.6: Measuring circulating IF116 in autoimmune patients and healthy
subjects using IFl16 ELISA with improved linearity.
The concentration of circulating IF116 in sera was determined by means of
ELISA in patients suffering from SSc (50), SLE (50), SjS (51), RA (50), anti-
phospholipid syndrome (pAPS, 80) and patients with hepatitis C virus
infection (HCV, 82) and in healthy subjects (CTRL, 50). The cohorts tested
represent different patients from those tested in Figure 3.
ELISA microtitre plates were coated with a polyclonal rabbit-anti-IF116
antibody. Subsequently, the plates were washed and free binding sites were
saturated with PBS/0,05%Tween-20/3%BSA (PBS-TB) at room temperature
for 1 hour. After washing, an incubation followed (1 h, room temperature)
with 5 pl of different sera samples in a final volume of 100 pl of
PBS/0,05%Tween-20/1 %BSA (PBS-TD). Purified 6His-IF116 protein, diluted
in 5% FBS in PBS-TD was used as standard. BSA served as negative
control. The samples were washed and in each case monoclonal mouse
anti-IF116 antibody was added and incubated for 1 h at room temperature.
After washing, an incubation followed (1 h, room temperature) with HRP-
conjugated anti-mouse antibody diluted in PBS-TD. After washing, the IF116
protein/antibody complex was visualised by incubation with
tetramethylbenzidine (TMB) and stopped with Stop Solution. The absorption
was measured at 450 nm in the micro plate reader. The determination of the
concentration was carried out using the standard curve of Fig. 5 for which
increasing concentrations of purified 6His-IF116 were used. The linearity of
the measurement ranged from 20 to 640 ng/ml IF116 in the sera. Sera with a
concentration outside the linearity range (<20 ng/ml or >640 ng/ml) are
plotted as having 0.1 ng/ml or 640 ng/ml respectively. The single grey
horizontal lines represent the mean IF116 concentrations for each group.

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-17-
A cut-off value for IF116 positivity was set at 95 percentile of control
population (117 ng/ml), and is represented by the light grey continuous
horizontal line. The numbers below the X axis represent the percentage of
patients with IF116 serum concentrations higher than the cut-off level in each
group. The IF116 serum protein was detectable at level higher than the cut-
off in a fraction of SSc, SLE, SjS, RA and HCV patients sera ranging from
20% to 80%, while only in 6% the healthy subjects. Only 1% of patients
suffering from pAPS were positive for circulating IF116.
Fig.7: Identification of extracellular IF116 in supernatants of cells
undergoing
cell death.
Human Keratinocyte monolayers were UVB-irradiated at different doses
(200, 400, and 800 J/m2 respectively), treated with 2pM Doxorubicin (Doxo)
and 8OpM Etoposide (VP-16), or left untreated. At 16h after treatment, the
supernatant was collected and separated for determination of extracellular
IFI16, while the remaining cells were lysed for determination of the
intracellular cleaved form of PARP (determination of undergoing cell death).
Collected supernatants were concentrated with TCA 25% as described in
Example 4. An equal amount of total cellular protein per sample and an
equal volume of concentrated supernatants were fractionated on SDS-PAGE
(7,5% of NEXT GEL Amresco, OH, USA) and transferred to a nitrocellulose
membrane (Biorad, CA, USA). Immunoblotting analysis using anti-IF116
polyclonal antibodies revealed the presence of extracellular IF116 in
supernatants of cells exposed to UVB irradiation doses of 400 and 800 J/m2.
This phenomenon is not generally associated with cell damage, because it
was not observed in keratinocytes undergoing chemically-induced cell death
upon exposure to pharmacological cytotoxic drugs as Doxorubicin and
Etoposide (as demonstrated by PARP cleavage, a recognized marker of
necrotic and apoptotic cell death (Cepeda V. et al., Recent Pat Anticancer
Drug Discov. 2006 Jan;1(1):39-53)).

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-18-
EXAMPLE II
IF116 ELISA
The following buffers were used for the IF116 ELISA developed: PBS-T
(0.25% Triton X100 in PBS); and PBS-TB (0.25% Triton X100 and 3% BSA
in PBS).
96-well ELISA plates (Nunc-Maxisorb Plates) were coated with 100 pl/well
anti-IF116 polyclonal antibody (incubation at 4 C for 16 h). The plates were
washed with PBS-T and blocked with PBS-TB for at least 30 min at room
temperature. The wells were aspirated and incubated as duplicates for 1 h at
37 C temperature with 100 pi of the standard (6His- IF116), diluted in a 5%
FBS in PBS-TB, or with 100 p1 of a sample at a suitable dilution (diluted with
PBS-TB), respectively. The wells were washed four times with PBS-T and
incubated for 1 h at 37 C with 100 pi of a monoclonal mouse antibody
against IF116, diluted in PBS-TB. Subsequently, the wells were washed 3
times with PBS-T and incubated with 100 pl of a peroxidase (HRP) which is
conjugated to a rabbit anti-mouse antibody (GE HealthCare, USA), diluted
1:500 in PBS-TB, for 1 h at 37 C. Subsequently, the wells were washed 3
times with PBS-T and were incubated with 100 pl tetra-methylbenzidine
(SureBlue-TMB, KPL, USA) and then stopped with 100 pl of stop solution
(TMB StopSolution, KPL, USA). The absorption was determined at 450 nm
in a micro plate reader (Tecan), with 620 nm as a reference. The
concentration of IFI16 in the sample was calculated by means of the
standard curve. The method showed a linearity of 1 to 15,6 ng/ml of
IF116/well. The variability of the results in the different assays was 9,7%
(inter-assay CV%=9,7%).

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
_19-
EXAMPLE 2
Western Blot analysis of IF116 in cell supernatants
Supernatants of human primary keratinocytes cultured in serum-free medium
(Epilife, Cascade Biologics, USA) were subjected to precipitation with tri-
chloroacetic acid (TCA). Precipitated protein were analyzed by immunoblot-
ting.
Cells were cultured in serum-free medium (Epilife, Cascade Biologics, USA)
and then exposed to different doses (from 200 to 800 J/m2) of ultraviolet B ir-
radiation (UVB) or mock-irradiated. 16 and 24 hours after irradiation, super-
natants were collected and centrifuged at 5000g for 10 minutes to remove
cellular debris. TCA was then added to supernatants at a final concentration
of 25% v/v, samples were incubated 10 min on ice and centrifuged at 4 at
14000g for 10 min. The protein pellet was washed 3 times with 100% acet-
one, air dried and resuspended in Laemmli Sample Buffer. Following denat-
uration at 950 for 5 min, the samples were loaded on 7.5% polyacrylamide
gel and subjected to gel electrophoresis.
Migrated proteins were transferred to nitrocellulose. The membrane was
blocked in TBS-5% BSA and extracellular IF116 was detected by membrane
incubation overnight at 4 C with anti-IF116 rabbit polyclonal antibody. After
3
washes with TBS-0.05%-Tween20 (TBS-T), the membrane was incubated
with a HRP-conjugated anti-rabbit secondary antibody (GE Healthcare, USA)
for 1 hour at room temperature. After washing with TBS-T, the membrane
was incubated with ECL (GE HealtCare) and the chemiluminescent signals
acquired by GelDoc image analyzer (BioRad, USA).

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-20-
EXAMPLE 3
IF116 ELISA with improved linearity
The following buffers were used for the IFI16 ELISA developed: PBS-TB
(0.05% Tween-20 and 3% BSA in PBS) and PBS-TD (0.05% Tween-20 and
1 % BSA in PBS).
96-well ELISA plates (Nunc-Maxisorb Plates) were coated with 100 pl/well
anti-IF116 polyclonal antiantibody (incubation at 4 C for 16 h). The plates
were washed with the wash buffer (Wash Solution Concentrate, KPL, USA)
and blocked with PBS-TB for at least 1 hour at room temperature. The wells
were washed and incubated as duplicates for 1 h at room temperature with
100 pl of the standard (6His- IF116), diluted in a 5% FBS in PBS-TD, or with
100 pl of a sample at a suitable dilution (diluted with PBS-TD), respectively.
The wells were washed and incubated for 1 h at room temperature with 100
pl of a monoclonal mouse antibody against IF116, diluted in PBS-TD.
Subsequently, the wells were washed and incubated with 100 pl of a
peroxidase (HRP) which is conjugated to a rabbit anti-mouse antibody (GE
HealthCare, USA), diluted 1:500 in PBS-TD, for 1 h at room temperature.
Subsequently, the wells were washed and incubated with 100 pl tetra-
methylbenzidine (SureBlue-TMB, KPL, USA) and then stopped with 100 pl of
stop solution (0.6N H2SO4). The absorption was determined at 450 nm in a
micro plate reader (Tecan), with 620 nm as a reference. The concentration
of IF116 in the sample was calculated by means of the standard curve. The
method showed a linearity of 1 to 32 ng/ml of IF116/well. A cut-off value was
set at the 95 percentile of the control population. Subjects displaying IF116
concentrations higher than the cut-off value were considered positive for the
presence of circulating IF116.
35

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-21-
EXAMPLE 4
Western Blot analysis of IF116 in supernatants of cells undergoing cell
death
Supernatants of human primary keratinocytes cultured in serum-free medium
(Epilife, Cascade Biologics, USA) were subjected to precipitation with tri-
chloroacetic acid (TCA). Precipitated protein were analyzed by immunoblot-
ting.
Cells were cultured in serum-free medium (Epilife, Cascade Biologics, USA)
and then exposed to different doses (from 200 to 800 J/m2) of ultraviolet B ir-
radiation (UVB) or treated with 2pM Doxorubicin (Doxo) or 80pM Etoposide
(VP-16) or mock-irradiated. At 16 hours after treatement, supernatants were
collected and centrifuged at 5000g for 10 minutes to remove cellular debris.
TCA was then added to supernatants at a final concentration of 25% v/v,
samples were incubated 10 min on ice and centrifuged at 4 at 14000g for
10 min. The protein pellet was washed 3 times with 100% acetone, air dried
and resuspended in Laemmli Sample Buffer.
As control of the undergoing cell death induced by the treatment with UVB-ir-
radiation and Doxo or VP-16, also the intracellular PARP cleavage was de-
termined in the exposed keratinocyte. For this proposed the Adherent cells
were lysed in RIPA buffer (50 mM Tris-cl pH 7.4, 150 mM NaCl, 1% NP40,
0.25% Na-deoxycholate, 1 mM PMSF, 1X complete mini protease inhibitor
cocktail (Roche), 1X phosphatase inhibitor cocktail (Pierce)).
Following denaturation at 950 for 5 min, the samples were loaded on 7.5%
polyacrylamide gel and subjected to gel electrophoresis.
Migrated proteins were transferred to nitrocellulose. The membranes were
blocked in TBS/0.05%Tween20/5%BSA and extracellular IF116 was detected

CA 02746288 2011-06-08
WO 2010/066913 PCT/EP2009/067128
-22-
by incubation of the membrane bearing surnatants samples with anti-IF116
mouse monoclonal antibody (clone 1G7, Santa Cruz, CA, USA). The intra-
cellular cleaved form of PARP was detected by incubation of the membrane
bearing cell extract samples by rabbit anti- PARP cleaved antibody
(GTX24830, GeneTex, CA, USA). After 3 washes with TBS/0.05%Tween20
(TBS-T), the membranes were incubated with a HRP-conjugated anti-mouse
or anti-rabbit secondary antibody (GE Healthcare, USA) respectively for 1
hour at room temperature. After washing with TBS-T, the membranes were
incubated with ECL (GE HealtCare) and the chemiluminescent signals ac-
quired by GelDoc image analyzer (BioRad, USA).

Representative Drawing
A single figure which represents the drawing illustrating the invention.
Administrative Status

2024-08-01:As part of the Next Generation Patents (NGP) transition, the Canadian Patents Database (CPD) now contains a more detailed Event History, which replicates the Event Log of our new back-office solution.

Please note that "Inactive:" events refers to events no longer in use in our new back-office solution.

For a clearer understanding of the status of the application/patent presented on this page, the site Disclaimer , as well as the definitions for Patent , Event History , Maintenance Fee  and Payment History  should be consulted.

Event History

Description Date
Time Limit for Reversal Expired 2015-12-15
Application Not Reinstated by Deadline 2015-12-15
Deemed Abandoned - Failure to Respond to Maintenance Fee Notice 2014-12-15
Inactive: Abandon-RFE+Late fee unpaid-Correspondence sent 2014-12-15
Inactive: Cover page published 2011-08-05
Inactive: Notice - National entry - No RFE 2011-07-28
Application Received - PCT 2011-07-28
Inactive: First IPC assigned 2011-07-28
Inactive: IPC assigned 2011-07-28
National Entry Requirements Determined Compliant 2011-06-08
Application Published (Open to Public Inspection) 2010-06-17

Abandonment History

Abandonment Date Reason Reinstatement Date
2014-12-15

Maintenance Fee

The last payment was received on 2013-11-06

Note : If the full payment has not been received on or before the date indicated, a further fee may be required which may be one of the following

  • the reinstatement fee;
  • the late payment fee; or
  • additional fee to reverse deemed expiry.

Patent fees are adjusted on the 1st of January every year. The amounts above are the current amounts if received by December 31 of the current year.
Please refer to the CIPO Patent Fees web page to see all current fee amounts.

Fee History

Fee Type Anniversary Year Due Date Paid Date
MF (application, 2nd anniv.) - standard 02 2011-12-14 2011-06-08
Basic national fee - standard 2011-06-08
MF (application, 3rd anniv.) - standard 03 2012-12-14 2012-11-15
MF (application, 4th anniv.) - standard 04 2013-12-16 2013-11-06
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
NOTOPHARM SRL
Past Owners on Record
ERICA MIRAGLIA
FRANCESCA GUGLIESI
MARISA GARIGLIO
MICHELE MONDINI
SANTO LANDOLFO
SILVIA COSTA
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
Documents

To view selected files, please enter reCAPTCHA code :



To view images, click a link in the Document Description column (Temporarily unavailable). To download the documents, select one or more checkboxes in the first column and then click the "Download Selected in PDF format (Zip Archive)" or the "Download Selected as Single PDF" button.

List of published and non-published patent-specific documents on the CPD .

If you have any difficulty accessing content, you can call the Client Service Centre at 1-866-997-1936 or send them an e-mail at CIPO Client Service Centre.


Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Description 2011-06-07 22 1,162
Claims 2011-06-07 2 68
Abstract 2011-06-07 2 58
Drawings 2011-06-07 7 214
Representative drawing 2011-08-04 1 8
Cover Page 2011-08-04 1 33
Notice of National Entry 2011-07-27 1 194
Reminder - Request for Examination 2014-08-17 1 117
Courtesy - Abandonment Letter (Request for Examination) 2015-02-08 1 164
Courtesy - Abandonment Letter (Maintenance Fee) 2015-02-08 1 174
PCT 2011-06-07 9 371